Integrative spatial analysis reveals a multi-layered organization of glioblastoma.

Glioma contains malignant cells in diverse states. Here, we combine spatial transcriptomics, spatial proteomics, and computational approaches to define glioma cellular states and uncover their organization. We find three prominent modes of organization. First, gliomas are composed of small local environments, each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. This pairing of states is consistent across tumors. Third, these pairwise interactions collectively define a global architecture composed of five layers. Hypoxia appears to drive the layers, as it is associated with a long-range organization that includes all cancer cell states. Accordingly, tumor regions distant from any hypoxic/necrotic foci and tumors that lack hypoxia such as low-grade IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of cellular states in glioma, highlighting hypoxia as a long-range tissue organizer.

LGR5 expressing skin fibroblasts define a major cellular hub perturbed in scleroderma.

Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5+-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies.

BCR affinity differentially regulates colonization of the subepithelial dome and infiltration into germinal centers within Peyer's patches.

Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer's patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.

Complete human day 14 post-implantation embryo models from naive ES cells.

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

Thymic mimetic cells function beyond self-tolerance.

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.

Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.

Decoupling cell size homeostasis in diatoms from the geometrical constraints of the silica cell wall.

Unicellular organisms are known to exert tight control over their cell size. In the case of diatoms, abundant eukaryotic microalgae, two opposing notions are widely accepted. On the one hand, the rigid silica cell wall that forms inside the parental cell is thought to enforce geometrical reduction of the cell size. On the other hand, numerous exceptions cast doubt on the generality of this model. Here, we monitored clonal cultures of the diatom Stephanopyxis turris for up to 2 yr, recording the sizes of thousands of cells, in order to follow the distribution of cell sizes in the population. Our results show that S. turris cultures above a certain size threshold undergo a gradual size reduction, in accordance with the postulated geometrical driving force. However, once the cell size reaches a lower threshold, it fluctuates around a constant size using the inherent elasticity of cell wall elements. These results reconcile the disparate observations on cell size regulation in diatoms by showing two distinct behaviors, reduction and homeostasis. The geometrical size reduction is the dominant driving force for large cells, but smaller cells have the flexibility to re-adjust the size of their new cell walls.

TP53 missense mutations in PDAC are associated with enhanced fibrosis and an immunosuppressive microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, which is refractory to all currently available treatments and bears dismal prognosis. About 70% of all PDAC cases harbor mutations in the TP53 tumor suppressor gene. Many of those are missense mutations, resulting in abundant production of mutant p53 (mutp53) protein in the cancer cells. Analysis of human PDAC patient data from The Cancer Genome Atlas (TCGA) revealed a negative association between the presence of missense mutp53 and infiltration of CD8+ T cells into the tumor. Moreover, CD8+ T cell infiltration was negatively correlated with the expression of fibrosis-associated genes. Importantly, silencing of endogenous mutp53 in KPC cells, derived from mouse PDAC tumors driven by mutant Kras and mutp53, down-regulated fibrosis and elevated CD8+ T cell infiltration in the tumors arising upon orthotopic injection of these cells into the pancreas of syngeneic mice. Moreover, the tumors generated by mutp53-silenced KPC cells were markedly smaller than those elicited by mutp53-proficient control KPC cells. Altogether, our findings suggest that missense p53 mutations may contribute to worse PDAC prognosis by promoting a more vigorous fibrotic tumor microenvironment and impeding the ability of the immune system to eliminate the cancer cells.

Energy Transport in Dichroic Metallo-organic Crystals: Selective Inclusion of Spatially Resolved Arrays of Donor and Acceptor Dyes in Different Nanochannels.

In this study, the precise positioning and alignment of arrays of two different guest molecules in a crystalline host matrix has been engineered and resulted in new optically active materials. Sub-nm differences in the diameters of two types of 1D channels are sufficient for size-selective inclusion of dyes. Energy transport occurs between the arrays of different dyes that are included in parallel-positioned nanochannels by Förster resonance energy transfer (FRET). The color of individual micro-sized crystals are dependent on their relative position under polarized light. This angular-dependent behavior is a result of the geometrically constrained orientation of the dyes by the crystallographic packing of the host matrix and is concentration dependent.

Bone morphology is regulated modularly by global and regional genetic programs.

Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9+/Scx+ superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview.

Characterization and possible function of an enigmatic reflector in the eye of the shrimp Litopenaeus vannamei.

Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 µm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.

Fibroblast recruitment as a tool for ovarian cancer detection and targeted therapy.

Metastatic ovarian cancer, the most lethal of gynecologic malignancies, is typically managed by debulking surgery, followed by chemotherapy. However, despite significant efforts, survival rate remains low. We have previously demonstrated, in mouse models, a specific systemic homing of labeled fibroblasts to solid ovarian tumors. Here, we demonstrate the feasibility of utilizing this specific homing of genetically modified fibroblasts for detection and targeted therapy of orthotopic metastatic ovarian carcinoma model in immune-deficient mice. Using an in vivo metastatic mouse model for ovarian cancer, we demonstrated that fibroblasts expressing fluorescent reporters injected intra-peritoneally, were specifically recruited to peritoneal tumor nodules (resulting in 93-100% co-localization). We further used fibroblasts over expressing the soluble receptor variant of VEGFR1 (s-Flt1). Mice bearing tumors were injected weekly with either control or s-Flt1 expressing fibroblasts. Injection of s-Flt1 expressing fibroblasts resulted in a significant reduction in the ascites volume, reduced vascularization of adherent metastases, and improved overall survival. Using fluorescently labeled fibroblasts for tumor detection with readily available intra-operative fluorescence imaging tools may be useful for tumor staging and directing biopsies or surgical efforts during exploratory or debulking surgery. Fibroblasts may serve as a beacon pointing to the otherwise invisible metastases in the peritoneal cavity of ovarian cancer patients. Utilizing the recruited fibroblasts also for targeted delivery of anti angiogenic or antitumor molecules may aid in controlling tumor progression. Thus, these results suggest a novel approach for targeting ovarian tumor metastases for both tumor detection and therapy.

Ovarian carcinoma: quantitative biexponential MR imaging relaxometry reveals the dynamic recruitment of ferritin-expressing fibroblasts to the angiogenic rim of tumors.

PURPOSE: To quantitatively monitor the dynamic perivascular recruitment of ferritin heavy chain (FHC)-overexpressing fibroblasts to ovarian carcinoma xenografts by using R2 mapping and biexponential magnetic resonance (MR) relaxometry. MATERIALS AND METHODS: In vivo studies of female mice were approved by the institutional animal care and use committee. In vitro analysis included MR-based R2 relaxation measurements of monkey kidney cell line (CV1) fibroblasts that overexpress FHC, followed by inductively coupled plasma mass spectrometry to assess cellular iron content. For in vivo analysis, CV1-FHC fibroblasts were either mixed with fluorescent human ovarian carcinoma cells before subcutaneous implantation (coinjection) or injected intraperitoneally 4 days after the cancer cells were injected (remote recruitment). Dynamic changes in tumor R2 were used to derive CV1-FHC cell fraction in both models. In coinjection tumors, dynamic contrast material-enhanced MR imaging was used to measure tumor fractional blood volume. Whole-body fluorescence imaging and immunohistochemical staining were performed to validate MR results. One-way repeated measures analysis of variance was used to assess MR and fluorescence imaging results and tumor volume, and one-way analysis of variance was used to assess spectrometric results, fractional blood volume, and immunohistochemical evaluation. RESULTS: CV1-FHC fibroblasts (vs CV1 fibroblasts) showed enhanced iron uptake (1.8 mmol ± 0.5 × 10(-8) vs 0.9 mmol ± 0.5 × 10(-8); P < .05), retention (1.6 mmol ± 0.5 × 10(-8) vs 0.5 mmol ± 0.5 × 10(-8), P < .05), and cell density-dependent R2 contrast. R2 mapping in vivo revealed preferential recruitment of CV1-FHC cells to the tumor rim in both models. Measurement of fractional blood volume was similar in all tumors (2.6 AU ± 0.5 × 10(-3) for CV1, 2.3 AU ± 0.3 × 10(-3) for CV1-FHC, 2.9 ± 0.3 × 10(-3) for CV1-FHC-ferric citrate). Dynamic changes in CV1-FHC cell fraction determined at MR relaxometry in both models were confirmed at immunohistochemical analysis. CONCLUSION: FHC overexpression, when combined with R2 mapping and MR relaxometry, enabled in vivo detection of the dynamic recruitment of exogenously administered fibroblasts to the vasculature of solid tumors.

Actin-dependent astrocytic infiltration is a key step for axon defasciculation during remodeling.

Astrocytes are essential for synapse formation, maturation, and plasticity; however, their function during developmental neuronal remodeling is largely unknown. To identify astrocytic molecules required for axon pruning of mushroom body (MB) γ neurons in Drosophila, we profiled astrocytes before (larva) and after (adult) remodeling. Focusing on genes enriched in larval astrocytes, we identified 12 astrocytic genes that are required for axon pruning, including the F-actin regulators Actin-related protein 2/3 complex, subunit 1 (Arpc1) and formin3 (form3). Interestingly, perturbing astrocytic actin dynamics does not affect their gross morphology, migration, or transforming growth factor ß (TGF-ß) secretion. In contrast, actin dynamics is required for astrocyte infiltration into the axon bundle at the onset of pruning. Remarkably, decreasing axonal adhesion facilitates infiltration by Arpc1 knockdown (KD) astrocytes and promotes axon pruning. Conversely, increased axonal adhesion reduces lobe infiltration by wild-type (WT) astrocytes. Together, our findings suggest that actin-dependent astrocytic infiltration is a key step in axon pruning, thus promoting our understanding of neuron-glia interactions during remodeling.

Exocytosis of the silicified cell wall of diatoms involves extensive membrane disintegration.

Diatoms are unicellular algae characterized by silica cell walls. These silica elements are known to be formed intracellularly in membrane-bound silica deposition vesicles and exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements remains unknown. Here we study the membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during its formation, the mineral phase is in tight association with the silica deposition vesicle membranes, which form a precise mold of the delicate geometrical patterns. We find that during exocytosis, the distal silica deposition vesicle membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. We demonstrate that within the cell, the proximal silica deposition vesicle membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.

The petrous bone contains high concentrations of osteocytes: One possible reason why ancient DNA is better preserved in this bone.

The characterization of ancient DNA in fossil bones is providing invaluable information on the genetics of past human and other animal populations. These studies have been aided enormously by the discovery that ancient DNA is relatively well preserved in the petrous bone compared to most other bones. The reasons for this better preservation are however not well understood. Here we examine the hypothesis that one reason for better DNA preservation in the petrous bone is that fresh petrous bone contains more DNA than other bones. We therefore determined the concentrations of osteocyte cells occluded inside lacunae within the petrous bone and compared these concentrations to other bones from the domestic pig using high resolution microCT. We show that the concentrations of osteocyte lacunae in the inner layer of the pig petrous bone adjacent to the otic chamber are about three times higher (around 95,000 lacunae per mm3) than in the mastoid of the temporal bone (around 28,000 lacunae per mm3), as well as the cortical bone of the femur (around 27,000 lacunae per mm3). The sizes and shapes of the lacuna in the inner layer of the petrous bone are similar to those in the femur. We also show that the pig petrous bone lacunae do contain osteocytes using a histological stain for DNA. We therefore confirm and significantly expand upon previous observations of osteocytic lacuna concentrations in the petrous bone, supporting the notion that one possible reason for better preservation of ancient DNA in the petrous bone is that this bone initially contains at least three times more DNA than other bones. Thus during diagenesis more DNA is likely to be preserved in the petrous bone compared to other bones.

DestVI identifies continuums of cell types in spatial transcriptomics data.

Most spatial transcriptomics technologies are limited by their resolution, with spot sizes larger than that of a single cell. Although joint analysis with single-cell RNA sequencing can alleviate this problem, current methods are limited to assessing discrete cell types, revealing the proportion of cell types inside each spot. To identify continuous variation of the transcriptome within cells of the same type, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI). Using simulations, we demonstrate that DestVI outperforms existing methods for estimating gene expression for every cell type inside every spot. Applied to a study of infected lymph nodes and of a mouse tumor model, DestVI provides high-resolution, accurate spatial characterization of the cellular organization of these tissues and identifies cell-type-specific changes in gene expression between different tissue regions or between conditions. DestVI is available as part of the open-source software package scvi-tools ( https://scvi-tools.org ).

ICAM-1 on Breast Cancer Cells Suppresses Lung Metastasis but Is Dispensable for Tumor Growth and Killing by Cytotoxic T Cells.

Breast tumors and their derived circulating cancer cells express the leukocyte ß2 integrin ligand Intercellular adhesion molecule 1 (ICAM-1). We found that elevated ICAM-1 expression in breast cancer cells results in a favorable outcome and prolonged survival of breast cancer patients. We therefore assessed the direct in vivo contribution of ICAM-1 expressed by breast cancer cells to breast tumorigenesis and lung metastasis in syngeneic immunocompetent mice hosts using spontaneous and experimental models of the lung metastasis of the C57BL/6-derived E0771 cell line, a luminal B breast cancer subtype. Notably, the presence of ICAM-1 on E0771 did not alter tumor growth or the leukocyte composition in the tumor microenvironment. Interestingly, the elimination of Tregs led to the rapid killing of primary tumor cells independently of tumor ICAM-1 expression. The in vivo elimination of a primary E0771 tumor expressing the ovalbumin (OVA) model neoantigen by the OVA-specific OVA-tcr-I mice (OT-I) transgenic cytotoxic T lymphocytes (CTLs) also took place normally in the absence of ICAM-1 expression by E0771 breast cancer target cells. The whole lung imaging of these cells by light sheet microscopy (LSM) revealed that both Wild type (WT)- and ICAM-1-deficient E0771 cells were equally disseminated from resected tumors and accumulated inside the lung vasculature at similar magnitudes. ICAM-1-deficient breast cancer cells developed, however, much larger metastatic lesions than their control counterparts. Strikingly, the vast majority of these cells gave rise to intravascular tumor colonies both in spontaneous and experimental metastasis models. In the latter model, ICAM-1 expressing E0771- but not their ICAM-1-deficient counterparts were highly susceptible to elimination by neutrophils adoptively transferred from E0771 tumor-bearing donor mice. Ex vivo, neutrophils derived from tumor-bearing mice also killed cultured E0771 cells via ICAM-1-dependent interactions. Collectively, our results are a first indication that ICAM-1 expressed by metastatic breast cancer cells that expand inside the lung vasculature is involved in innate rather than in adaptive cancer cell killing. This is also a first indication that the breast tumor expression of ICAM-1 is not required for CTL-mediated killing but can function as a suppressor of intravascular breast cancer metastasis to lungs.